We have expressed two truncated isoforms of chicken nonmuscle myosin II- B using the Baculovirus expression system. One of the expressed heavy meromyosins (HMM-exp) consists of a 150 kD myosin heavy chain (MHC), comprising amino acids 1-1231 as well as the 20 kD and 17 kD myosin light chains (MLCs) in a 1:1:1 molar ratio. The second HMM-exp was identical except that it contained an insert of 10 amino acids (PESPKPVKHQ) at the 25-50 kD domain boundary in the subfragment-1 region of the MHC. Expressed HMMs were soluble at low ionic strength, bound to rabbit skeletal muscle actin in an ATP-dependent manner and these properties afforded a rapid purification of mg quantities of expressed protein. Both isoforms were capable of moving actin filaments in the in vitro motility assay and manifested a greater than 20-fold activation of actin-activated MgATPase activity following phosphorylation of the 20 kD MLC. mRNA encoding the isoform containing the 10 amino acid insert in MHC II-B has been detected only in neuronal tissues in chickens and mammals (Takahashi, M., Kawamoto, S., and Adelstein, R.S., J. Biol. Chem. 267, 17864, 1992; Itoh, K., and Adelstein, R.S., ibid 270, 14533, 1995). Inserted HMM-exp was phosphorylated by cdc2, cdK5 and MAP kinase in vitro to 0.3-0.4 mols Pi/mol MHC. The site phosphorylated in the MHC was identified as the serine residue present in the 10 amino acid insert. Characterization of the noninserted, inserted and phosphorylated MHC isoforms with respect to actin-activated MgATPase activity and ability to translocate actin filaments in an in vitro motility assay produced the following average values: Noninserted HMM-exp, Vmax = 0.28 s-1, Km = 12.7 uM; Translocation rate = 0.077 um/sec; Inserted HMM-exp, Vmax = 0.37 s-1, Km = 15.1 uM; Translocation rate = 0.092. HMM-exp that had been phosphorylated by p34-cdc2 kinase to 0.3 mols PO4/mol MHC showed a 30% increase in Vmax and a 20% increase in rate of actin translocation.